Prostaglandin modulation of the mechanism of ACTH action in the human adrenal

Vol. 73, No. 1, 1976

BIOCHEMICAL

AND BIOPHYSICAL

PROSTAGLANDIN MODULATION

RESEARCH COMMUNICATIONS

OF THE MECHANISM OF ACTH

ACTION IN THE HUMAN ADRENAL Kenneth V. Honn and Walter Chavin Departments of Radiology and Biology Wayne State University, Detroit, Michigan 48202, U.S.A. Received

July

26,1976

Summary: PGEl and PGE2 significantly increased human adrenal CAMP levels in vitro; cortisol output was also increased in a dose related fashion. In contrast, PGFla and PGFz~ depressed adrenal CAMP (except PGFz~ at 100 ug/ml) . PGFla and PGF2, depressed cortisol levels at all doses. Indomethacin or 7-oxa- 13-prostynoic acid did not affect these parameters. However, when applied in conjunction with ACTH they inhibited or enhanced hormonal action depending upon the temporal sequence of application. The findings indicate that prostaglandins modulate ACTH-adrenocortical cell interaction bidirectionally, initially potentiating and subsequently depressing ACTH stimulated events.

Prostaglandins have been demonstrated to stimulate adrenal steroidoInitially,

genesis in a number of mammalian species. occur primarily via pituitary effects of prostaglandins

such was believed to

ACTH release ( 1) , but subsequently the direct

on adrenocortical

CAMP (2-4) and steroidogenesis

(2,4- 6) were demonstrated. The demonstration of prostaglandin receptors in human and ovine adrenal cell plasma membranes (3) provide direct support to the effects of exogenous prostaglandins

in adrenal physiology.

glandin biosynthesis

ACTH induced E and F type prosta-

from 3H-arachidonic

acid (7) affirm the concept that

prostaglandins are a link in the ACTH stimulation of steroidogenesis . However, it is unclear whether prostaglandins function in addition to and/or as an integral part of ACTH-adrenocortical

cell interaction.

The present study was

designed to further illuminate the actions of several prostaglandins CAMP and steroidogenesis.

Evidence is presented for bidirectional

on adrenal prosta-

glandin modulation of human adrenal function. MATERIAL AND METHODS Six adult human female adrenal glands obtained at surgery were immediately placed in cold (0-4OC) Kreb’s Ringer bicarbonate buffer, KRBGA (pH 7.4, 164 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. 73, No. 1, 1976

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200mg glucose/dl, 0.5% serum albumin fraction V) . Glands were diced { 2x3 mm and preincubated (37oC) in KRBGA for 45 min. These dice then were incubated (lml KRBGA; 37OC; 95%02+ 5%co2) in a Dubnoff metabolic shaker for 1-32 min. The dice were exposed to prostaglandins El, E2, Fla or F2a at 1 , 10 ,100 ug/ml , prostaglandin vehicle ( 2%ethanol in KRBGA), indomethacin ( 10 ug/ml) , EC- I- 148 ( 7- oxa- 13- prostynoic acid; 50 ug/ml) , porcine ACTH ( 100 mIU/ml; chromatographically pure: 150 IU/mg) or KRBGA alone. Further, dice were incubated initially ( 4 min) in ACTH, indomethacin or EC- I- 148 followed by transfer to ACTH plus the appropriate test substance. Adrenal incubates were quenched in liquid nitrogen and analyzed for CAMP by RIA (8). Cortisol secretion into the incubation medium was quantitated by RIA ( 9) . Proteins were determined (10) and the data expressed as pM CAMP or ng cortisol/mg protein, &SEM . A minimum of four replicates were used per datum point. Data were analyzed by analysis of variance and student t test. Differences were accepted as significant when p ( 0.05. Table 1. --In Vitro Effects of Prostaglandins El, E2, Fla and F2a Upon Human Adrenocortical CAMP Levels. Incubation Intervals Represent The Earliest Times At Which Maximal Stimulation Or Depression Of CAMP Levels Were Observed. Treatment

Incubation Interval (min)

PM CAMP /mg protein (Mean + SEM)

2

KRBGA

5.32

0.2

8

KRBGA

4.42

1.4

16

KRBGA

4.32

2.0

2

PGEl

1 ug/ml

IO.92

3.5”

2

PGEl

10 ug/ml

17.82

2.5*

2

PGE1

100 ug/ml

33.02

6.6*

8

PGE2

1 ug/ml

7.92

3.6

8

PGE2

10 ug/ml

7.52

4.0

8

PGE2

100 ug/ml

38.02

7.4*

8

PGFla

1 ug/ml

8

PGFla

10 ug/ml

8

PGFla 100 ug/ml

undetectable

a

PGF2, 100 ug/ml

21.0~

16

PGF2a

16

PGF2,

*Statistically

different

1.2 + 0.8* 2.6~

1.0

5.8*

1 ug/ml

1.82

0.8

10 ug/ml

2.12

0.5

from corresponding control values.

165

Vol. 73, No. 1, 1976

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

f 600 1

1Cl

P%

100 up/ml

600

C

Fig 1

PGE ,

PGEz

“GFla

--In vitro dose related prostaglandin stimulation ( PGEl , PGE2) and depression (PGFla, PGF2,) of cortisol output (32 min) relative to controls (C) by human adrenocortical tissue.

INWMETHACIN

o’,,, 124

-

AClH

+ INCOMETHACIN

,

,

I

8

16

24

TIME,

Fig 2

PGha

I’ 32

min.

Effects of preincubation (4 min) in either 7-oxa- 13-prostynoic acid (EC-I-148) or indomethacin on the subsequent CAMP response to ACTH (100 mIU/ml) compared to the control CAMP response to this ACTH dose in human adrenocortical tissue.

RESULTS PGEl (1 ,lO,lOO

us/ml) significantly

response occurring within 2 min (Table 1). levels

increased CAMP levels with a peak PGEz significantly

increased CAMP

(8 min) at 100 ug/ml; 1,lO ug/ml increased CAMP levels (8 min) 80% and

70% respectively significantly

but were not statistically

depressed CAMP levels

significant.

PGFla (1 ug/ml)

(8 min) below controls while 10 ug/ml

depressed CAMP levels (8 min) 59% and 100 ug/ml resulted in undetectable CAMP levels

(Table 1). Although not statistically

significant,

PGF2,

(1,lO ug/ml) decreased CAMP levels (16 min) 58% and 51%, respectively.

166

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Vol. 73, No. 1, 1976

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

0 124

6

16 TIME,

Fig 3

24

32

min.

Supramaximal CAMP response of human adrenocortical tissue with indomethacin or 7- oxa- 13-prostynoic acid (EC- I- 148) in ACTH following preincubation (4 min) in ACTH ( 100 mIU/ml).

P 600 2-400 -I p 200 88 0 / A

Fig 4

Interestingly

Cortisol output by human adrenocortical tissue following 32 min incubation in: A. KRBGA B. 7- oxa- 13- prostynoic acid C. indomethacin D. ACTH E. ACTH following 7- oxa- 13-prostynoic acid preincubation F. ACTH following indomethacin preincubation G. 7-oxa- 13-prostynoic acid following ACTH preincubation H. indomethacin following ACTH preincubation

PGF3, ( 100 us/ml) significantly

above controls (Table 1).

increased CAMP levels (8 min)

PGEl and PGE2 significantly

increased cortisol in a

dose related manner, whereas PGFla and PGF2, significantly cortisol output at all doses studied (Fig 1). alterations

from the control incubations

depressed

The vehicle did not produce any

in KRBGA alone in the two parameters

evaluated.

167

Vol. 73, No. 1,1976

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AND BIOPHYSICAL

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Neither indomethacin or EC- I- 148 significantly However,

pretreatment

affected CAMP levels.

(4 min) with either test substance followed by incuba-

tion in ACTH and the test substance resulted in a significant

depression of

CAMP levels below that produced by ACTH at 2- 16 min (Fig 2), Alternatively, pretreatment

(4 min) with ACTH followed by incubation in ACTH and either test

substance (Fig 3) resulted in an heightened CAMP response at 4-8 min (indomethacin) or 16-32 min (EC-I- 148). significantly

Neither indomethacin or EC-I- 148 alone

altered cortisol output from that of the controls (Fig 4).

adrenocortical

cell suspensions,

this indomethacin concentration

however,

show augmented corticoid

output at

( 11) . Preincubation in either test substance

followed by ACTH and the test substance significantly put below control and ACTH levels.

depressed cortisol out-

Cortisol secretion after ACTH preincu-

bation followed by ACTH and test substance was significantly alone but significantly

Cat

higher (indomethacin)

lower than ACTH

than the controls (Fig 4).

DISCUSSION The human adrenal is stimulated by the E series prostaglandins to increase both CAMP levels and cortisol findings in the lower forms studied. activation

of adenylate cyclase,

output.

This agrees with the basic

The CAMP response appears mediated via

but alternate effects of the prostaglandins

upon phosphodiesterases , protein kinases, ATPase, etc. also resulting in increased CAMP levels should not be overlooked

(12).

portant factor in the understanding of adrenocortical actions.

Time may be an im-

cell prostaglandin inter-

The present data indicate that PGEl evoked the maximal CAMP re-

sponse with considerable rapidity

(2 min) compared to ACTH (8 min) . The de-

creased CAMP response time suggests the possibility

of direct activation

of

adenylate cyclase by this prostaglandin to achieve more than a six fold increase in the cyclic

nucleotide.

PGE2, on the other hand achieved the CAMP peak

response at (8 min) thereby suggesting that alternate mechanisms may be operative in this instance.

In addition,

both E series prostaglandins stimulated

cortisol release on a similar temporal basis despite the temporally dissimilar CAMP response. for significant

As a detectable

corticosteroid

ated events affecting

CAMP increase is not always a prerequisite

release (13) the possibility

of prostaglandin medi-

steroidogenesis without the intervention

consideration. 168

of CAMP merits

BIOCHEMICAL

Vol. 73, No. 1,1976

AND BIOPHYSICAL

PGFla and PGF2, act in a manner antagonistic prostaglandins sol output.

RESEARCH COMMUNICATIONS

to that of the E type

in that the former decrease both human adrenal CAMP and corti-

An inverse relationship

between the E and F type prostaglandins

also has been suggested in other systems (11).

An interesting

exception to the

aformentioned results is the stimulation of human adrenal CAMP by high levels (100 ug/ml) of PGF2,. E and F prostaglandin

This finding is not inconsistent are antagonistic,

observed to nonspecifically ing results characteristic

with the hypothesis that

for high levels of PGF2, have been

cross react with E prostaglandin receptors producof that group ( 12).

ug/ml) increased human adrenocortical

Notably,

CAMP levels,

although PGF2a i 100 corticoid

output was de-

pressed, a result consistent with the other doses of F prostaglandins tested. Clearly the prostaglandin

mediated CAMP and corticosteroid

considered separate events,

alterations

The stimulation of adrenocortical

can be

production of

both E and F type prostaglandins by ACTH (7) would appear to be paradoxical the two prostaglandin types have opposing effects.

However,

experiments which combine ACTH with a prostaglandin or antagonist

(7-oxa- 13- prostynoic

of the interaction

cortical

considering our

inhibitor

(indomethacin)

acid) it is clear that the temporal sequence

produces either CAMP inhibition

s ponse . Therefore,

or a heightened CAMP re-

both sets of experiments suggest that the ACTH-adreno-

cell interaction

may be modulated by prostaglandins in a bidirectional

fashion in that prostaglandins may initially

potentiate

subsequently depress that effect or vice versa.

the ACTH effect but

This may occur via a negative

feedback by a single type of prostaglandin or an antagonistic between prostaglandins

interaction

of the E and F series.

ACKNOWLEDGEMENTS The authors are grateful to Dr. J.E. Pike, Upjohn Co., for the prostaglandins and to Dr. J. Fried, University of Chicago,for the 7-oxa- 13-prostynoic acid.

REFERENCES 1. 2.

as

Peng, T.C., Six, K.M. and Munson, P.L. (1970) Endocrinology 86, 202-206 Warner, W. and Rubin, R.P. (1975) Prostaglandins2, 83-95

169

Vol. 73, No. 1, 1976

3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Dazord, A., Morera, A.M., Bertrand, J. and Saez, J.M. (1974) Endocrinology%, 352-359 Saruta, T. and Kaplan, N.M. (1972) J. Clin. Invest. 51, 2246-2251 Flack, J.D. and Ramwell, P.W. (1972) Endocrinology%, 371-377 Flack, J.D., Jessup, R., Ramwell, P.W. (1969) Science 163, 691-692 Laychock, S.G. and Rubin, R.P. (1975) Prostaglandins 10, 529-540 Steiner, A.L., Parker, C .W. and Kipnis, D.M . (1972) J. Biol. Chem. 247, 1106-1113 Foster, L.B. and Dunn, R.T. (1974) Clin. Chem. 2, 365-368 Honn, K.V. and Chavin, W. (1975) Anal. Biochem. 68, 230-235 Laychock, S.G. and Rubin, R.P. (1976) Brit. J. Pharmacol. 2, 273-278 Kuehl, F .A. ( 1974) Prostaglandins 2, 325-340 Beall, R. J. and Sayers, G. (1972) Arch. Biochem. Biophys. 148, 70-76

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